Evaluating bias-reducing protocols for RNA sequencing library preparation.
Evaluating bias-reducing protocols for RNA sequencing library preparation.
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DOI：
10.1186/1471-2164-15-569
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发表时间：
2014-07-07
期刊：
影响因子：
4.4
通讯作者：
Willis AE
中科院分区：
文献类型：
--
作者：
Jackson TJ;Spriggs RV;Burgoyne NJ;Jones C;Willis AE
下一代测序无法对读数丰度产生完全无偏的估计，这可能会影响从测序数据中得出的结论。 RNA 测序文库生成中的连接步骤是已知的偏差来源，推动了酶技术和文库构建方案的发展。我们首次将 Life Technologies 提供的用于 Ion Torrent PGM 的标准双工适配器协议与涉及 CircLigase（CircLig 协议）的替代单适配器方法进行比较。先前已报道过测序文库中的过度表达与二级结构程度之间的相关性，因此我们还研究了是否可以通过与在不允许这种结构的温度下起作用的酶连接来减少偏差。使用三种方案之一将一组已知组成的小 RNA 片段转化为测序文库，并在 Ion Torrent PGM 上进行测序。与标准方案相比，CircLig 方案减少了特定序列的过度表达。过度呈现的序列更有可能被预测为具有二级结构并与接头序列共折叠。然而，使用热稳定连接酶热自养甲烷杆菌 RNA 连接酶 K97A (Mth K97A) 不足以减少偏差。基于 CircLigase 的单适配器方法显着减少了 Ion Torrent 数据中的偏差，但并未消除偏差。在一定温度下起作用以消除二级结构对文库生成的可能影响的连接可能是有价值的，尽管 Mth K97A 在这种情况下无效。本文的在线版本 (doi:10.1186/1471-2164-15-569) 包含补充材料，可供授权用户使用。
Next-generation sequencing does not yield fully unbiased estimates for read abundance, which may impact on the conclusions that can be drawn from sequencing data. The ligation step in RNA sequencing library generation is a known source of bias, motivating developments in enzyme technology and library construction protocols. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig protocol). A correlation between over-representation in sequenced libraries and degree of secondary structure has been reported previously, therefore we also investigated whether bias could be reduced by ligation with an enzyme that functions at a temperature not permissive for such structure. A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The CircLig protocol resulted in less over-representation of specific sequences than the standard protocol. Over-represented sequences are more likely to be predicted to have secondary structure and to co-fold with adaptor sequences. However, use of the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) was not sufficient to reduce bias. The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Ligases that function at temperatures to remove the possible influence of secondary structure on library generation may be of value, although Mth K97A is not effective in this case. The online version of this article (doi:10.1186/1471-2164-15-569) contains supplementary material, which is available to authorized users.
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影响因子：
4.4
作者：
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DOI：
10.1186/1748-7188-6-26
发表时间：
2011-11-24
期刊：
Algorithms for molecular biology : AMB
影响因子：
--
作者：
Lorenz R;Bernhart SH;Höner Zu Siederdissen C;Tafer H;Flamm C;Stadler PF;Hofacker IL
通讯作者：
Hofacker IL